Journal: bioRxiv

Article Title: STANDARDISED FLOW CYTOMETRIC PROCEDURE FOR DEEP CHARACTERIZATION OF NANOPARTICLES

doi: 10.1101/2024.07.18.604065

Figure Lengend Snippet: Panels A-B: FCM analysis of 0.22 μm filtered PBS acquired with threshold applied on FITC fluorescent channel (band pass 525/40). Panels C-E: Megamix Plus FSC/SSC polystyrene beads are used to define the resolution limit of the instrument and to compare the efficiency in separating smallest beads on violet side scatter (V-SSC) compared to the conventional blue side scatter (B-SSC). Threshold was applied on FITC fluorescent channel. Panels G-H: FCM analysis of unstained EVs released by A375M melanoma cell line (Blank sEVs). Panels I-N: FCM analysis of fluorescent EVs released by A375M melanoma cell line pulsed with the green fluorescent fatty acid, BODIPY™ FL C16 (C16 EVs). Red boxes include beads between 100 nm and 200 nm in size.

Article Snippet: Instrument were aligned with Rainbow QC Beads (Spherotech), while the detector settings for nanovesicle detection and fine-tuning adjustments of the alignment were made with Megamix-Plus SSC/FSC beads (BioCytek).

Techniques:

Journal: bioRxiv

Article Title: STANDARDISED FLOW CYTOMETRIC PROCEDURE FOR DEEP CHARACTERIZATION OF NANOPARTICLES

doi: 10.1101/2024.07.18.604065

Figure Lengend Snippet: Representative density plots of reference beads (Megamix Plus FSC/SSC), analyzed applying the threshold on the fluorescence or on the V-SSC channels, are shown in A and B, respectively. PBS filtered at 0.22 µm was used to exclude the background noise (C, D). The black boxes were drawn to include the smallest sized beads (< 200 nm) and to exclude the background noise. The C16 sEVs that fall in the black box were enumerated (E, F). The numbers in red, inside the dot plots C, D, E, and F, indicate the events per µl.

Article Snippet: Instrument were aligned with Rainbow QC Beads (Spherotech), while the detector settings for nanovesicle detection and fine-tuning adjustments of the alignment were made with Megamix-Plus SSC/FSC beads (BioCytek).

Techniques: Fluorescence

Journal: bioRxiv

Article Title: STANDARDISED FLOW CYTOMETRIC PROCEDURE FOR DEEP CHARACTERIZATION OF NANOPARTICLES

doi: 10.1101/2024.07.18.604065

Figure Lengend Snippet: The B-SCC Area signal versus B-SCC Width signal allowed the exclusion of vesicle aggregates and background noise from the population of singlet particles (A). Representative dot plots and respective histograms of singlet gated Megamix-Plus FSC (B, C) and SSC (D, E) polystyrene beads with size between 100 nm and 900 mm (0.1µm, 0,16µm, 0,2µm, 0,24µm, 0.3 µm, 0.5 µm and 0.9 µm). The analysis was performed on the events falling in the singlet region (see black arrow).

Article Snippet: Instrument were aligned with Rainbow QC Beads (Spherotech), while the detector settings for nanovesicle detection and fine-tuning adjustments of the alignment were made with Megamix-Plus SSC/FSC beads (BioCytek).

Techniques:

Journal: Genes

Article Title: Extracellular Vesicles from NSC-34 MN-like Cells Transfected with Mutant SOD1 Modulate Inflammatory Status of Raw 264.7 Macrophages

doi: 10.3390/genes15060735

Figure Lengend Snippet: Quantitative analysis of large and small EVs isolated from mSOD1 NSC-34 MN-like cells using high-resolution flow cytometry. ( A ): Flow cytometry gating strategy for phenotyping large and small EVs. Megamix-Plus FSC is used to set FSC-vSSC for EV events. APC-Annexin V and tetraspanin APC-CD81 are used to stain isolated GFP-positive lEVs and sEVs, respectively. Before analysis on a CytoFLEX S flow cytometer, EVs are diluted 1:200 in 1X PBS. For all assays, 30 μL of the control (unstained EV) and 30 μL of each sample were measured at slow flow. ( B ), Histogram of GFP/Annexin V+ (lEVs) and GFP/CD81+ (sEVs) events in EV subtypes isolated from mSOD1 NSC-34. Graphs show mean ± SEM.

Article Snippet: Fluorescent Megamix-Plus SSC calibration beads (BioCytex, Marseille, France) were used to optimize cytometer settings and to define the EV gate.

Techniques: Isolation, Flow Cytometry, Staining, Control